This proposal seeks to pursue our investigations on the pathogenesis of amyotrophic lateral sclerosis (ALS) by searching for the earliest cellular perturbations provoked by mutant superoxide dismutase-1 (SOD1), a known cause of this fatal disease. This project rests on the premise that mutant SOD1 induces responses in genes and proteins expressed by motor neurons even before the emergence of the clinical phenotype which are key to understanding the basis of motor neuron degenerative process and to developing effective neuroprotective strategies. We have shown that wild-type primary or embryonic stem cell-derived spinal motor neurons can be killed by astrocytes expressing mutant SOD1, a neurotoxic effect that can be recapitulated with medium pre-conditioned by mutant, but not wild-type astrocytes (Nagai et al., Nat. Neurosci. 2007). Given these results, we hypothesize that our in vitro cell system should afford a unique tool to identify those initial mRNA and protein changes that arise in wild-type motor neurons in response to a mutant SOD1. Accordingly, in Specific Aim (SA)-I, we will determine motor neuron response to mutant astrocyte conditioned medium by gene array using highly purified primary spinal cord and embryonic stem cell-derived motor neurons cultured on laminin, and then, at a defined moment, exposed to either mutant or wild-type astrocyte conditioned medium. At selected time points, motor neurons cultured in mutant or wild-type astrocyte conditioned medium will be harvested, and total mRNA will be isolated and quantitatively compared by gene expression profiling. Results will be confirmed by real-time PCR. In SA-II, we will define motor neuron response to mutant astrocyte conditioned medium by proteomics using the same neuronal cultures and conditional media as in SA-I. At selected time points, motor neurons, cultured as above, will be harvested and proteins will be isolated and quantitatively compared by differential fluorescence 2-D gel electrophoresis proteome profiling. Results will be confirmed by Western blot. This R21 project offers a high-risk/high-yield set of studies, which may identify, thanks to complementary approaches, molecular pathways and key mediators of motor neuron degeneration in the mutant SOD1 model. The generated information may be of critical importance for the development of effective neuroprotective therapies for the familial form of ALS linked to mutant SOD1 and perhaps for its sporadic form as well. PUBLIC HEALTH RELEVANCE: Amyotrophic lateral sclerosis (ALS) is an incurable fatal paralytic disorder of uncertain cause. We have found in a dish that specific inflammatory cells isolated from animals modeling this disease produce factors capable of killing the nerve cells responsible for ALS paralysis. In this project, we propose to use this unique disease model to hunt for the earliest pathological changes taking place in nerve cells in response to the insult mediated by neighboring inflammatory cells. We expect that these findings will open the door to the development of new therapeutic strategies aimed at protecting nerve cells against ALS even before they become significantly damaged by the disease process.